ABSTRACT: Leptin is secreted by adipocytes in amounts indicative of body fat content. Variations in leptin levels contribute to hypothalamic regulation of feeding and metabolism to maintain body weight homeostasis. Importantly, if there is increasing leptin with increasing body fat and the hypothalamus cannot detect or respond to it, such as is seen in leptin resistance, obesity may develop. Growth hormone (GH) is secreted by the anterior pituitary and directly influences body composition through lipolysis. GH also stimulates the secretion of insulin-like growth factor I (IGF-1), which mediates some of the actions of GH. The hypotheses of this dissertation are (1) under normal circumstances, leptin stimulates GH, and (2) in the leptin resistance situation described above, leptin fails to stimulate GH. In obesity, GH is severely attenuated, and leptin resistance may be the mechanism by which this occurs. To test the first hypothesis, rats were given diets with varying fat contents and GH was measured. The diet with the highest fat content did not cause obesity; there were elevated levels of leptin but not enough to produce resistance. These animals had an enhanced GH-IGF-1-axis. In a second study, leptin treatment resulted in elevated GH secretion from a GH-secreting cell line (GH1), further strengthening the hypothesis. To test the second hypothesis, rats were given normal rat chow and implanted with osmotic minipumps for the continuous infusion of two doses of leptin or vehicle. The leptin-treated animals developed resistance, as measured by loss of effect on food intake but not on various metabolic measures, including glucose, triglycerides, and insulin. IGF-1 was attenuated in the high-dose leptin group. Leptin receptors were reduced in this study. A final study utilized both the diets of varying fat content and the leptin-filled osmotic minipumps. Leptin was shown to lose its effectiveness in animals fed the high-fat diet and IGF-1 was attenuated. The rats were fed the diets for a longer period than in the first diet study, allowing time for the development of leptin resistance. IGF-1 was also attenuated in animals infused with leptin. The results of these two studies agree with the second hypothesis.

conditions was made by rough estimation. Negative controls, including a sample lacking

the reverse transcription enzyme and a sample lacking RNA, were included (data not

shown).

? '2 A L : '7

1 2 3 4 5

.7 400

Figure 2-1: MgCl2 (mM) and temperature
(C) optimization
Lane 1 is the 100 base pair (bp) DNA ladder;
lane 2 is 0.5 mM at 630C; lane 3 is 1.0 mM at
630C; lane 4 is 1.5 mM at 630C; lane 5 is 2.0
mM at 630C; lane 6 is 0.5 mM at 640C; and lane
7 is 1.0 mM at 640C. Based on the results of this
experiment, the conditions used for the
subsequent studies were 1 mM MgCl2 at 640C.

Figure 2-2: Cycle optimization
Lane 1 is the mass DNA ladder; lane 2 is
25 cycles; lane 3 is 30 cycles; lane 4 is 35
cycles; and lane 5 is 40 cycles. Based on
the results of this experiment, 30 cycles
were used in subsequent experiments.

Southern Blot and Normalization

The identity of RT-PCR product (Figure 2-3) was confirmed using the internal probe 5'-

Each leptin receptor protein measured with this Western protocol gave bands of multiple
molecular weights. No pre-immune or pre-absorption samples were run, so the
specificity of the bands is uncertain. The molecular weight of the largest band is what
was expected for the leptin receptor, so it can be assumed to be specific. The lower
molecular weight bands have been seen in studies using different antibodies [Boes et al.
1999], so these are possibly specific as well.

compared to control (p<0.05). Similarly, the valley nadirs were higher in high-fat rats

and lower in low-fat rats, but these results were not significant. Control rats were in the

middle of the range for both peak height and valley nadir. As expected, because of

elevated pulse amplitudes, the high-fat rats spent less time in the GH trough periods

(valley width, p=0.01) compared to the other groups. Peak width, measured in minutes,

was not significantly different among the groups. Representative profiles for the high-fat

(panel A), control (panel B), and low-fat rats (panel C) are given in Figure 3-6.

0 10 20 30

Figure 3-1: Body Weight
There were significant differences in body weights between low- and high-fat rats (n=9) beginning on day
10 and remaining for most of the duration of the study and between the low-fat (n=8) and control rats (n=8)
on days 10-14 and 18-29 (2-way repeated measures ANOVA, p<0.05). Rats fed the high-fat diet gained
significantly more weight than controls beginning at day 18 and remaining for most of the duration of the
study.

B

* * * * **

5 10 15 20 23 30 35 0
Day

310 C

o t0

0 5 10 IS 20 30 35 40
Day

-- High-Fat Diet
Low-Fat Diet
Normal Chow

Figure 3-2: Food Intake
When measured in grams (g food), it was shown that rats fed the high-fat diet (n=9) consumed significantly
less chow than rats fed the control diet (n=8) beginning within the first 5 days of the study and remaining
for the duration (2-way repeated measures ANOVA, p<0.05, panel A). No comparisons for animals fed the
low-fat diet (n=8) are reported versus either of the other two groups because these rats were pair-fed in
grams of food to the high-fat rats. When measured in grams of fat consumed (panel B), the high-fat rats
consumed significantly more than the control rats, which in turn consumed significantly more than the low-
fat rats (2-way repeated measures ANOVA, p<0.05). When measured in kcal (panel C), normal chow rats
ate more for most of the study, but the values were nearly normalized.

16-

4
-1
0 *

Normal Chow Low-Fat High-Fat
Diet
Figure 3-3: Leptin Levels
At the end of the study, leptin levels were significantly elevated (1-way ANOVA, p<0.05) in the rats fed
the high-fat diet (n=8) compared to those fed either of the other two diets (n=6 each).

SA
S * *

0 5 t0 IS 2w 25 30 35 40

Control Low-Fat High-Fat

Diet

Figure 3-4: Ob-Rb mRNA
There were no significant differences (1-way ANOVA, p<0.05) leptin receptor mRNA in hypothalamus
due to any of the different diets (n=6 for each group). Leptin receptor was normalized using the
housekeeping gene cyclophilin.

1400

1200

1000

800

600

400

Normal Chow Low Fat High Fat

Diet
Figure 3-5: IGF
At the end of the study, IGF-1 levels were significantly elevated (1-way ANOVA, p<0.05) in the rats fed
the high-fat diet (n=8) compared to those fed either of the other two diets (n=6 each).

71

Table 3-1: Growth Hormone Profile of Diet-Treated and Control Rats

Overall

Group High-Fat Diet Low-Fat Diet Normal Chow p values

Total Area

Under Curve 22,249.60 4,494.65 + 12,439.97 p<0.10

(ng/mL) 3,359.90 1,099.25 4,412.45

Mean Area

Under Curve 60.65 12.62 34.74 + p<0.10

(ng/mL.min) 8.73 3.47 11.82

Peak Width 46.25 58.75 37.50

(minutes) 6.25 8.75 4.33 N/S

Peak Height 288.63 36.81 + 142.01

(ng/mL) 59.60 7.88 47.53 p<0.05

Valley Width 45.00 66.25 62.50

(minutes) 0.00 1.25 2.50 p=0.01

Valley Nadir 25.75 5.56 11.68

(ng/mL) 10.71 1.99 3.70 N/S

Values are given as mean SEM. High-fat and low-fat diet groups, n=2 each; controls,
n=3. Overall p values are given to indicate significance or trends. N/S means not
significant at p<0.10 level.

Time
Figure 4-3: Media Supplemented with 10% Horse Serum and 2.5% FBS
Significantly more GH was secreted at 24 hours (n=6) than at 8 hours (t-test, p<0.05, n=6). Leptin had no
effect at either 8 (n=6) or 24 hours (n=6).